Traditionally, in analysis of the primary structure of proteins and others, the Edman method has been used, in which a labeled amino acid such as 3-phenyl-2-thiohydantoin (PTH)amino acid is assayed by reverse phase high performance liquid chromatography. However, since this method is based on UV absorption, it is significantly affected by background factors such as the organic solvent of the eluent and decomposition products, and the sensitivity is insufficient.
In recent years, a microassay method based on fluorescence has been developed to meet the requirements for quicker analysis and higher precision. In this method, the amino group in amino acid is labeled with a fluorescent reagent, including an isothiocyanate derivative such as fluorescein isothiocyanate (FITC).
Usually, it is necessary to eliminate the excess reagent after labeling the fluorescent reagent; a problem has been pointed out that it is difficult to set conditions for elimination of the excess reagent in the case of FITC and other reagents having a hydrophilic functional group in their molecular structure.
When the compound to be detected is a compound whose polarity is different from that of the isothiocyanate derivative, the reaction solution may be isolated directly by chromatography after labeling with the fluorescent reagent. On the other hand, in the case of compounds having a hydrophilic functional group, such as amino acids and sugars, it is often difficult to separate the labeled derivative from the excess reagent. If this excess fluorescent reagent remains in the sample, a problem arises that the identification of amino acid derivatives is hampered in the detection of amino acids such as tryptophan and amino sugars such as glucosamine and galactosamine because the chromatographic peaks overlap or appear very closely.
On the other hand, if the excess reagent is completely washed out, the target amino acids etc. in the sample are flown out, which results in reduction in the sample volume.